Auxin Protectors Research Articles

Phloroglucinol in plant tissue culture

In plant tissue culture research, there is a constant need to search for novel substances that could result in better or more efficient growth in vitro. A relatively unknown compound, phloroglucinol (1,3,5-trihydroxybenzene), which is a degradation product of phloridzin, has growth-promoting properties. Phloroglucinol increases shoot formation and somatic embryogenesis in several horticultural and grain crops. When added to rooting media together with auxin, phloroglucinol further stimulates rooting, most likely because phloroglucinol and its homologues act as auxin synergists or auxin protectors. Of particular interest is the ability of phloroglucinol—a precursor in the lignin biosynthesis pathway—to effectively control hyperhydricity through the process of lignification, thus maximizing the multiplication rate of woody species and other species that are difficult to propagate. Phloroglucinol has also been used to improve the recovery of cryopreserved Dendrobium protocorms, increasing the potential of cryopreservation for application in ornamental biotechnology. Phloroglucinol demonstrates both cytokinin-like and auxin-like activity, much like thidiazuron, and thus has considerable potential for application in a wide range of plant tissue culture studies.

Root induction in three species of bamboo with different rooting abilities

Rooting of in vitro axillary shoots from adult field culms of Bambusa atra, Dendrocalamus giganteus and D. hookeri and of juvenile seedling shoots of D. giganteus were investigated. B. atra rooted spontaneously without exogenous auxin during axillary shoot proliferation, while both Dendrocalamus species rooted only on transfer to rooting media with indole-3-butyric acid (IBA). D. giganteus required coumarin, an auxin protector, in addition to IBA. Rooting in adult shoots of D. giganteus was lower (45.6%) than that in the juvenile shoots (96.7%) but adult shoots of D. hookeri rooted well (88.9%). A pretreatment with thidiazuron (TDZ: N-phenyl- N-[(1,2,3-thidiazol-5-yl)urea]) induced development of axillary buds and subsequently 95% rooting in adult D. giganteus shoots but had no significant effect on rooting in juvenile shoots of D. giganteus or D. hookeri. Pretreatment with tri iodobenzoic acid (TIBA) arrested shoot and bud development leading to very low or no rooting in all three species.

Effect of exogenous indole butyric acid on root formation and peroxidase and indole-3-acetic acid oxidase activities and phenolic contents in date Palm offshoots

Exogenous indole butyric acid had a significant positive effect on the rooting response of date Palm (Phoenix dactylifera L.) offshoots. IBA-treated offshoots rooted earlier and with a much higher frequency while untreated offshoots rooted poorly, later, and tended to produce fewer roots. The effect of exogenous application of auxin was also reflected in the metabolic changes in offshoots during the rooting process. Pox activity showed a minimum on the sixth day and a maximum on the twelfth day after the root-inducing treatment. Changes of IAA oxidase activity showed a parallel time curve while phenolic contents and auxin protector levels showed an inverse trend. These parameters changed slightly one week later in the untreated offshoots.

Effect of abscisic acid on endogenous IAA, auxin protector levels and peroxidase activity during adventitious root initiation in Vigna radiata cuttings

Endogenous levels of free and conjugated IAA, auxin protectors (Prs) and peroxidase (PER) activity and their relation to adventitious root initiation (ARI) were investigated at the potential sites of adventitious rooting in relation to exogenous application of 250 µM ABA during the first 120 h after treatment. Cuttings from 7-day-old mung bean [Vigna radiata (L.) Wilcz.] seedlings were treated with 125, 250, and 500 µM ABA for 24 h. ABA significantly stimulated ARI but extremely inhibited epicotyl growth as compared to control. Free and conjugated IAA were measured by reversed-phase high performance liquid chromatography while Prs and PER activities were measured spectrophotometrically. The present results also indicate that endogenous free IAA levels peaked later in ABA-treated cuttings than that in control, suggesting that ABA extended the length of the induction phase of rooting process in treated cuttings and that might explain the significant delay of the appearance of roots at the treated cuttings. Higher level of IAA conjugates was found in ABA-treated cuttings than that in untreated ones. Pr level also peaked later in ABA-treated cuttings than that in control, indicating that ABA extended the period of Pr activity. An initial temporary decrease of PER activity was found in associating with high levels of free IAA and Prs during most of the primary events, while the opposite occurred during the secondary events of adventitious rooting process in both treated and untreated cuttings. Thus, ABA may stimulate ARI in mung bean Vigna radiata cuttings by regulating the concentration and /or activities of endogenous IAA, Prs, and PER activity in favor of inducing a large number of adventitious roots at their potential sites of adventitious rooting.

IAA-oxidase activity and auxin protectors in nonrooting, rac, mutant shoots of tobacco in vitro

The peroxidase and IAA-oxidase activities, the degree of auxin protection and the amount of soluble phenolics were determined in in vitro cultured shoots of a nonrooting mutant, rac, of tobacco compared to its wild homologue. The mutant and wild shoots showed similar peroxidase variations along the growth cycle of 21 days, but with higher levels of activity for the rac mutant. During this growth cycle, the minimum of peroxidase activity occurred at day 14 for both tobacco whole shoots. However, this minimum of activity did not occur at the same day in the basal part of the stem, where roots may appear, of the two types of tobacco. Both mutant and wild whole shoots showed about the same IAA-oxidase activity in the fractions resulting from a gel filtration of the crude extracts through a Sephadex G-100 column but differed in the degree of auxin protection. The rac shoots exhibited a very high level of auxin protectors of low molecular weight, among which chlorogenic acid. They were also characterized by eight to nine times higher level of soluble phenolics. The relationships between these biochemical aspects in relation to the absence of root formation in the rac mutant are discussed.

Methyl gallate and related polyphenols as auxin protectors

The systematic study of the effect of chestnut polyphenols (gallic acid, methyl gallate, (+)-catechin, (−)-epicatechin, epicatechin gallate, epigallocatechin and epigallocatechin gallate) on indole-3-acetic acid (IAA)-oxidase activity, proved that the tested polyphenols are much less inhibitory than their gallates. The inhibition caused by the methyl ester of gallic acid at 2 μM was about eight times stronger than that of gallic acid itself.

Polyphenolic auxin protectors in buds of juvenile and adult chestnut

Polyphenolic auxin protectors in buds of juvenile and adult chestnut

Polypheolic auxin protectors in buds of juvenile and adult chestnut

Both qualitative and quantitative estimates of the relative amounts of several poly‐phenols in buds of juvenile and adult plants of chestnut (Castanea sativa x C. crenata) were carried out. In buds of both types of plants, chesnatin, crenatin, cretanin and (+)‐catechin were identified. Crenatin and cretanin showed inhibitory activity of peroxidase‐catalysed indoleacetic acid oxidation. Auxin protection capacity was greater in extracts of the juvenile tissues which also were richer in active phenols. The phenolic content and its possible relationship to the easier establishment in vitro of juvenile tissue is discussed.

Changes in levels of peroxidases and phenolics during root formation in Vitis cultured in vitro

Root formation in vine (Vitis vinifera L. cv. Albariño) was accompanied by at first a rise and then a fall in total peroxidase (EC 1.11.1.7) activity in the explant. These variations ran parallel to similar changes in the amount of the three cathodic isope‐roxidases detected, the most abundant of which, C2, also exhibited IAA oxidase activity. The anodic isoperoxidase bands detected were very weak and underwent no variation during rooting. Changes in the opposite direction (a fall followed by a rise) were shown by certain endogenous phenolics, including monoferuloyl, monocaffeoyl and mono‐ρ‐coumaroyl tartaric acids, some of which may act as auxin protectors.

Changes in auxin protectors and IAA oxidases during the rooting of chestnut shoots in vitro

Auxin protectors and IAA oxidase activity were comparatively analyzed in the upper and the lower parts of shoots of chestnut (Castanea sativa Mill.) cultivated in vitro with indolebutyric acid (IBA) pretreatment. Rhizogenesis of the shoots is accompanied by an increase in auxin protectors in the lower parts and by a decrease of these protectors in the upper parts. Besides, the IAA oxidase activity declines in the basal parts during the rooting process while it increases in the upper ones. These biochemical events would enhance the IAA level in the rooting region of the shoots. In untreated, non‐rooted cuttings, the IAA oxidase activity remains low in the upper parts and high in the basal parts of the shoots. The results thus indicate that the IBA treatment may control the endogenous auxin level of the cuttings, either through a direct regulation of the IAA oxidase system or more indirectly through the transport of auxin protectors.

Association of Auxin Protectors, Peroxidase, Indoleacetic Acid Oxidase and Polyphenol Oxidase in Zizyphus Gall and Normal Stem Tissues Grown in Culture

Summary Zizyphus normal stem tissue extract destroyed IAA when the two were incubated together. On the other hand, with gall tissue extract, IAA destruction occurred only following a lag period which was induced by auxin protectors detected in the gall tissue. The in vivo gall tissue showed no peroxidase and little IAA-oxidase activity for the incubation period tested. Polyphenol oxidase activity and total and O-dihydroxyphenols increased with the growth of the gall up to the 30th day. The protector-induced lag in IAA destruction was decreased when higher concentrations of IAA were used in the reaction mixture. Both normal and gall tissues were exposed to effectors such as NAA, IAA, 2,4-D, DL-tryptophan, gibberellic acid, and cycloheximide, each added separately. A differential response of normal and gall tissues to the different growth regulators in terms of activities of peroxidase, IAA oxidase and polyphenol oxidase was esthablishcd. A general tendency towards a decrease in polyphenol oxidase and an increase in peroxidase and IAA oxidase activities was observed in gall tissue. Both in vivo and in vitro gall tissue showed a higher protein content as compared to normal tissue.

Changes in Soluble and Bound Peroxidase—IAA Oxidase During Tomato Fruit Development

ABSTRACTSoluble peroxidase activity increased dramatically during the early stages of tomato fruit development, reaching a maximum at the mature‐green stage. Ionically and covalently bound peroxidases were also observed, and the activities of these fractions increased steadily throughout fruit development. IAA (indoleacetic acid) oxidase activity was observed in both soluble and bound fractions and paralleled the peroxidase activity. Tissue homogenates from juvenile fruit caused an extended induction period in IAA oxidation reactions catalyzed by the purified tomato fruit peroxidase, suggesting the occurence of a high concentration of phenolic‐type auxin protectors in this tissue. As the fruit developed, tissue homogenates showed a reduction in the amount of auxin protectors as the IAA oxidizing capacity of the fruit increased.

Habituation in sugarbeet callus: auxin content, auxin protectors, peroxidase pattern and inhibitors

A GC‐ECD titration of IAA in normal auxin (2,4‐D)‐requiring and auxin‐independent (habituated) sugarbeet callus revealed an equal amount in both tissues. A comparison of the content and pattern (through starch gel electrophoresis) of soluble, membrane and wall peroxidases indicated that normal tissues contained a higher level of isoperoxidases. Normal tissues also were found to contain higher levels of peroxidase inhibitors and auxin protectors. The hypothesized peroxidase‐mediated higher rate of auxin destruction in normal sugarbeet callus is supposed to be counterbalanced by the 2,4‐D‐controlled auxin protectors.

Organogenesis in Habituated Sugarbeet Callus: Auxin Content and Protectors, Peroxidase Pattern and Inhibitors

Summary A comparison of the soluble, membrane- and cell wall-bound peroxidases of an organogenetic and a nonorganogenetic habituated sugarbeet callus showed that organogenetic tissues contained a higher level of peroxidase activity and a larger number of peroxidase isoenzymes. Partially (through G-100 Sephadex) purified peroxidases of organogenetic callus likewise exhibited higher auxin-oxidase activities. Organogenetic tissues also were found to contain higher levels of peroxidase inhibitors and auxin protectors (auxin-oxidase inhibitors). A GC-MS analysis of IAA indicated a lower level of the hormone in the organogenetic callus. This presumably leads to an auxin-cytokinin ratio compatible with the induction of shoot formation.

Presence of auxin protectors inEriophyes inducedZizyphus stem galls

3 auxin protectors, which were o-dihydroxyphenols of high molecular weight, were isolated fromZizyphus gall tissues. An increase in polyphenol oxidase activity in the gall tissue was observed which probably led to the production of high levels of auxin protectors. This prevented IAA oxidation, resulting in hyperauxinity and auxin-autotrophy.

Studies on auxin protectors XIV. Chlorogenic acid, a low molecular weight auxin protector in sunflower

Abstract Acetone extracts of sunflower leaves yielded two low molecular weight compounds with significant auxin protector activity. By means of chromatography, UV spectra, and acid hydrolysis it was possible to ascertain that most of the protector activity was associated with chlorogenic acid. Some protector activity was also associated with a derivative of chlorogenic acid, tentatively identified as isochlorogenic acid. A possible role for chlorogenic acid is proposed.

The masking of peroxidase-catalysed oxidation of IAA in Vigna

Partially purified enzyme preparations of extracts of Vigna seedlings exhibited guaiacol-oxidase activity but not IAA-oxidase activity. However, by ageing the enzyme preparations, or by treating them with H 2O 2, it was possible to unmask IAA-oxidase activity. Gel filtration of Vigna extracts on Sepharose yielded separate peaks for IAA-oxidase, guaiacol-oxidase and auxin protectors. The appearance of a separate IAA-oxidase peak reflected the overlap of peroxidase and protector; the apparent difference in the migration rate of IAA-oxidase and guaiacol-oxidase activity proved to be an artifact. The data imply that previous reports of differences between peroxidase and IAA oxidase need to be reinvestigated to rule out the possible effect of contamination by endogenous, high MW auxin protectors. A rapid method for removing most of the auxin protectors and thereby unmasking IAA-oxidase activity is described.

Soluble and cell-wall peroxidases and auxin destruction in normal and habituated tobacco callus

A comparison of the soluble and cell-wall-bound isoperoxidases of normal auxin-requiring and auxin-independent (habituated) tobacco callus revealed that normal tissues contained a higher level of isoperoxidases. There were also qualitative differences in these isoperoxidases. Partially purified soluble and ionically bound isoperoxidases of normal callus likewise exhibited higher auxin-oxidase activities. Normal tissues also were found to contain higher levels of auxin-oxidase inhibitors (auxin protectors). Overall, however, the data indicate that there is a higher rate of auxin destruction in normal tobacco callus than in habituated tissue. This presumably leads to insufficient endogenous auxin for growth.

Isolation and partial characterization of auxin protectors from Synchytrium endobioticum incited tumors in potato

Auxin protectors were isolated from potato tubers, potato sprouts and Synchytrium endobioticum incited tumors (potato wart). Three groups of protector were distinguished by molecular sieve chromatography. Dormant tubers contained relatively low levels of auxin protector compared to sprouts and tumor tissue. Tumors differed from normal tissues by their relatively high content of high molecular weight protector. The water-soluble high molecular weight protector from tumors was partially purified and shown to be a protein fraction (mol. wt 10 000 to 20 000) containing covalently bound caffeic and ferulic acids. The protein fraction contained unusually high levels of serine (15·4%), glycine (14·5%), glutamic acid (14·1%), proline (9·7%), aspartic acid (9·1%) and alanine (7·3%). A water-insoluble high molecular weight protector fraction was also isolated from wart tissue and shown to also contain covalently bound caffeic and ferulic acids but to differ from the water soluble fraction in its unusually high content of hydroxyproline (18%). It is suggested that cell wall proteins may be an important source of protector activity in potato wart tissue.

Studies on Auxin Protectors XII. Auxin Protectors and Polyphenol Oxidase in Developing Coffee Fruit

AbstractThe fruit of the coffee plant (Coffea arabica) was analyzed for auxin protector content. Ripe coffee berries were separated into pit and pulp, ground in buffer, and assayed for auxin protectors. The extracts were then subjected to gel filtration in order to determine the molecular weight of the protector(s). In the pit, a single protector was found with a molecular weight approaching 5000 daltons, while the pulp contained several auxin protectors, the largest of which appeared to be about 1000 daltons. Chromato‐graphic studies of various gel filtration fractions showed that protector activity was always associated with spots which exhibited a light blue fluorescence under UV.The changing patterns during coffee fruit development were also investigated. Auxin protector production, and polyphenol oxidase (E.C. 1.10.3.1), an enzyme related to protector metabolism, were assayed at weekly intervals. In the unripe berry, an auxin protector was found with a molecular weight exceeding 200,000 daltons; as the berry ripened the amount of this protector gradually decreased until almost none was present in the ripe berry and the pattern changed to the pattern described above. Polyphenol oxidase content decreased as the berry ripened.Commercially roasted pits, i.e., coffee “beans”, contained very high levels of protector activity. However, gel filtration studies showed this activity to be associated entirely with low molecular weight compounds.